RESUMO
OBJECTIVE: Sirt6, reactive oxygen species and ferroptosis may participate in the pathogenesis of Diabetic Nephropathy (DN). Exploring the relationship between Sirt6, oxidative stress, and ferroptosis provides new scientific ideas to DN. METHODS: Human podocytes were stimulated with 30 mM glucose and 5.5 mM glucose. The mice of db/db group were randomly divided into two groups:12 weeks and 16 weeks. Collect mouse blood and urine specimens and renal cortices for investigations. HE, Masson, PAS and immunohistochemical staining were used to observe pathological changes. Western blot, RT-qPCR and immunofluorescence staining were used to evaluate expression of relevant molecules. CCK8 method was introduced to observe cell viability. The changes of podocyte mitochondrial membrane potential and mitochondrial morphology in each group were determined by JC-1 staining and Mito-Tracker. RESULTS: The expression level of Sirt6, Nrf2, SLC7A11, HO1, SOD2 and GPX4 were reduced, while ACSL4 was increased in DN. Blood glucose, BUN, Scr, TG, T-CHO and 24h urine protein were upregulated, while ALB was reduced in diabetic group. The treatment of Ferrostatin-1 significantly improved these changes, which proved ferroptosis was involved in the development of DN. Overexpression of Sirt6 might ameliorate the oxidation irritable reaction and ferroptosis. Sirt6 plasmid transfection increased mitochondrial membrane potential and protected morphology and structure of mitochondria. The application of Sirt6 siRNA could aggravated the damage manifestations. CONCLUSION: High glucose stimulation could decrease the antioxidant capacity and increase formation of ROS and lipid peroxidation. Sirt6 might alleviate HG-induced mitochondrial dysfunction, podocyte injury and ferroptosis through regulating Nrf2/GPX4 pathway.
Assuntos
Nefropatias Diabéticas , Ferroptose , Fator 2 Relacionado a NF-E2 , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Podócitos , Sirtuínas , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Sirtuínas/metabolismo , Sirtuínas/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Camundongos , Animais , Humanos , Podócitos/metabolismo , Podócitos/patologia , Masculino , Estresse Oxidativo , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , Progressão da DoençaRESUMO
Nutrition therapy is the cornerstone treatment for chronic kidney disease (CKD). Although much attention has been given to dietary protein intake in CKD patients, many findings now demonstrate that the type of dietary protein intake may be more critical for CKD patients. In protein bioavailability and malnutrition prevention, many physicians recommend that CKD patients adhere to a low protein diet and restrict their plant foods, such as vegetables, fruits, and soybeans. However, nephrologists should not ignore the potential benefits of plant foods for CKD patients. It is not advisable to restrict the intake of plant foods in the later stage of CKD simply to prevent the development of hyperkalemia and malnutrition. This article highlights the benefits and possible problems of a plant-dominant low protein diet (PLADO) diet, defined as an LPD with dietary protein intake of 0.6-0.8 g/kg/day with at least 50% plant-based source for CKD patients. We hope to provide new opinions for clinical work and CKD patients.
Assuntos
Desnutrição , Insuficiência Renal Crônica , Animais , Humanos , Progressão da Doença , Proteínas Alimentares , Dieta com Restrição de ProteínasRESUMO
Aims: There are limited studies on phase angle and sarcopenia in continuous ambulatory peritoneal dialysis patients. So, we want to explore the association between phase angle and sarcopenia and find a more sensitive indicator for diagnosing sarcopenia. Methods: We included 101 continuous ambulatory peritoneal dialysis patients from March 2022 to August 2022 and measured the phase angle and body composition by bioelectrical impedance analysis. All patients had their handgrip strength measured. Then, we divided patients into the sarcopenia (n = 30) group and non-sarcopenia (n = 71) group according to the sarcopenia diagnostic strategy formulated by the Asian Working Group for Sarcopenia. We used logistic regression to explore the risk factors of sarcopenia. We applied Receiver-operating characteristics curves to determine the diagnostic accuracy of these risk factors. Results: After adjustments for sex, age, diabetes, BMI, extracellular water ratio, extra water, serum creatinine, total kt/v, and residual kt/v, phase angle correlated to handgrip strength and lowered limb muscle mass but not to skeletal muscle mass, upper arm muscle circumference, upper limb muscle mass and appendicular skeletal muscle mass index. In the multivariate logistic model, low phase angle and older age are risk factors for sarcopenia. The AUROC of phase angle for sarcopenia is 0.79 (95%CI, 0.70-0.86, P < 0.01) for both sexes, 0.70 and 0.85 for females and males. After we combined age and phase angle as diagnostic indicators of sarcopenia, the AUROC is 0.91 (95%CI, 0.83-0.96, P < 0.0001) in both sexes, 0.89 and 0.93 for females and males. Conclusion: This study illustrates that age 52 or older is an independent risk factor for sarcopenia in continuous ambulatory peritoneal dialysis patients. Phase angle can act as a predictor of sarcopenia in those patients. But the combination of age and phase angle is more valuable in diagnosing sarcopenia.
RESUMO
Background: Ubiquitin-52 amino acid fusion protein (UbA52) is an important factor in the pathogenesis of diabetic kidney disease (DKD) and has been suggested a potential marker in the disease. However, whether upregulation of UbA52 marks early kidney injury in T2DM mellitus (T2DM) patients remains unclear. In this study, we examine the diagnostic value of UbA52 as a biomarker in predicting early diabetic kidney disease (DKD) in T2DM patients. Methods: We used two-step ELISA to test UbA52 level in urine of 3 defined patient groups. Samples from T2DM patients without albuminuria or diabetic retinopathy (DM-WNP; n=30), T2DM patients with albuminuria and diabetic retinopathy, excluding other renal diseases clinically (DM-NP; n=30) and healthy controls (n=30) were analyzed. Spearman's correlation analysis and multiple linear regression model were used to analyze the correlation of urinary UbA52 level with laboratory results regarding kidney function. Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of UbA52 in predicting T2DM and early DKD. Results: Urinary UbA52 level in DM-NP group was 1.75 times and 2.71 times higher than in DN-WNP (p=0.004) and normal control group (p<0.001), respectively. The level of urinary UbA52 correlated significantly with serum creatinine (r=0.468, p<0.001), GFR (r=−0.300, p=0.004) and proteinuria (r=0.484, p<0.001). Multiple linear regression analysis showed that proteinuria level was independently associated with urinary UbA52 level (β=0.833, p<0.001). The area under the ROC of urinary UbA52 in diagnosing T2DM and DKD was 0.751 and 0.755, respectively. (AU)
Antecedentes: La proteína de fusión de aminoácidos ubiquitina-52 (UbA52) es un factor importante en la patogénesis de la enfermedad renal diabética (ERD), y se ha sugerido como marcador potencial en la enfermedad. Sin embargo, aún no está claro si la regulación al alza de UbA52 indica una lesión renal temprana en pacientes con diabetes mellitus de tipo 2 (DMT2). En este estudio, analizamos el valor diagnóstico de UbA52 como biomarcador para predecir la ERD temprana en pacientes con DMT2. Métodos: Utilizamos un ELISA de 2 pasos para analizar el nivel de UbA52 en la orina de 3 grupos de pacientes definidos. Se analizaron muestras de pacientes con DMT2 sin albuminuria o retinopatía diabética (DM-WNP; n=30), pacientes con DMT2 y con albuminuria y retinopatía diabética, excluyendo clínicamente otras enfermedades renales (DM-NP; n=30) y controles sanos (n=30). Se utilizó el análisis de correlación de Spearman y el modelo de regresión lineal múltiple para analizar la correlación del nivel de UbA52 en orina con los resultados de laboratorio relativos a la función renal. Se utilizó la curva de características operativas del receptor (ROC) para evaluar el valor diagnóstico de UbA52 en la predicción de la DMT2 y de la ERD temprana. Resultados: El nivel de UbA52 en orina en el grupo DM-NP fue 1,75 y 2,71 veces mayor que en el grupo DN-WNP (p=0,004), y en el grupo de control normal (p<0,001), respectivamente. El nivel de UbA52 en orina se correlacionó significativamente con la creatinina sérica (r=0,468; p<0,001), la TFG (r=−0,300; p=0,004) y la proteinuria (r=0,484; p<0,001). El análisis de regresión lineal múltiple mostró que el nivel de proteinuria se asociaba de forma independiente al nivel de UbA52 en orina (β=0,833; p<0,001). El área bajo las ROC de UbA52 en orina en el diagnóstico de la DMT2 y de la ERD fue de 0,751 y 0,755, respectivamente. (AU)
Assuntos
Humanos , Ubiquitinas , Nefropatias/diagnóstico , Diabetes Mellitus Tipo 2 , Ensaio de Imunoadsorção Enzimática , Nefropatias Diabéticas , Albuminúria , ProteinúriaRESUMO
BACKGROUND: Ubiquitin-52 amino acid fusion protein (UbA52) is an important factor in the pathogenesis of diabetic kidney disease (DKD) and has been suggested a potential marker in the disease. However, whether upregulation of UbA52 marks early kidney injury in T2DM mellitus (T2DM) patients remains unclear. In this study, we examine the diagnostic value of UbA52 as a biomarker in predicting early diabetic kidney disease (DKD) in T2DM patients. METHODS: We used two-step ELISA to test UbA52 level in urine of 3 defined patient groups. Samples from T2DM patients without albuminuria or diabetic retinopathy (DM-WNP; n=30), T2DM patients with albuminuria and diabetic retinopathy, excluding other renal diseases clinically (DM-NP; n=30) and healthy controls (n=30) were analyzed. Spearman's correlation analysis and multiple linear regression model were used to analyze the correlation of urinary UbA52 level with laboratory results regarding kidney function. Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of UbA52 in predicting T2DM and early DKD. RESULTS: Urinary UbA52 level in DM-NP group was 1.75 times and 2.71 times higher than in DN-WNP (p=0.004) and normal control group (p<0.001), respectively. The level of urinary UbA52 correlated significantly with serum creatinine (r=0.468, p<0.001), GFR (r=-0.300, p=0.004) and proteinuria (r=0.484, p<0.001). Multiple linear regression analysis showed that proteinuria level was independently associated with urinary UbA52 level (ß=0.833, p<0.001). The area under the ROC of urinary UbA52 in diagnosing T2DM and DKD was 0.751 and 0.755, respectively. CONCLUSION: The level of urinary UbA52 increased significantly in T2DM patients with DKD. The level of proteinuria is independently associated with urinary UbA52 level. Urinary UbA52 could serve as an early marker in the diagnosis of DKD. CLINICALTRIALS: gov Identifier: NCT02204280.
Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Retinopatia Diabética , Albuminúria/complicações , Aminoácidos , Biomarcadores , Creatinina/urina , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/etiologia , Humanos , UbiquitinasRESUMO
Effect of continuous use of dexmedetomidine during general anesthesia on perioperative levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in patients undergoing radical resection of ovarian cancer was investigated. The initial treatment of ovarian cancer is mainly radical surgery. Most patients with ovarian cancer radical surgery can achieve good results, but the use of improper anesthetic drugs in radical surgery can easily lead to unstable patient vital signs. Therefore, the selection of appropriate anesthetic drugs has become the key to radical ovarian cancer surgery. There are few reports on the use of dexmedetomidine in anesthesia for ovarian cancer radical surgery. This study was performed to retrospectively analyze the case data of patients undergoing laparoscopic ovarian cancer radical surgery, and to compare the hemodynamics of dexmedetomidine anesthesia with midazolam anesthesia and the concentrations of TNF-α and IL-6, to provide reference for clinical implementation of ovarian cancer radical surgery. The hemodynamics of patients in the dexmedetomidine group were stable compared with the midazolam group. Serum TNF-α and IL-6 levels were significantly lower in the dexmedetomidine group than that in the midazolam group. If dexmedetomidine were continuously used during general anesthesia, the perioperative serum levels of TNF-α and IL-6 could be effectively reduced in patients undergoing radical resection of ovarian cancer, and the perioperative stress response was suppressed.
RESUMO
The aim of the present study was to investigate the effects of transcription factor EB (TFEB) overexpression on oxidative stress, mitochondrial function and apoptosis in podocytes induced with high glucose. High glucoseinduced timedependent changes in TFEB expression were identified and nuclear translocation of TFEB was observed in podocytes. Overexpression of TFEB markedly reduced high glucoseinduced oxidative stress in podocytes, and increased the expression of superoxide dismutase 2 and heme oxygenase 1 antioxidant enzymes. It was further observed that TFEB overexpression could partially restore the expression of peroxisome proliferatoractivated receptorγ coactivator1α, transcription factor A, mitochondrial, and cytochrome c oxidase subunit 4, thereby enhancing mitochondrial biosynthesis. Furthermore, overexpression of TFEB reduced mitochondrial swelling and fragmentation, restored mitochondrial membrane potential, and contributed to the restoration of mitochondrial function. By overexpressing TFEB, it was revealed that TFEB increased the ratios of phosphorylated (p)Akt/Akt and pBad/Bad, and the expression of downstream Bclxl, and reduced the ratio of Bax/Bcl2 and the expression of cleavedcaspase3 compared with high glucosetreatment. Furthermore, when the Akt phosphorylation inhibitor Ly294002 was added, the improvement by TFEB to high glucoseinduced apoptosis was significantly reduced. These findings suggest that overexpressing TFEB could reduce the production of reactive oxygen species in podocytes in a high glucose environment, relieve oxidative stress, promote mitochondrial biogenesis and renewal functions, and reduce high glucoseinduced podocyte apoptosis by activating the Akt/Bad pathway.
Assuntos
Apoptose , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Glucose/metabolismo , Estresse Oxidativo , Podócitos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Podócitos/citologia , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaAssuntos
Glomerulonefrite Membranosa , Rim , Neoplasias , Síndromes Paraneoplásicas/imunologia , Trombospondinas/imunologia , Idoso , Autoantígenos , Biomarcadores , Feminino , Glomerulonefrite Membranosa/diagnóstico , Glomerulonefrite Membranosa/etiologia , Glomerulonefrite Membranosa/imunologia , Humanos , Imuno-Histoquímica , Rim/diagnóstico por imagem , Rim/imunologia , Rim/patologia , Imageamento por Ressonância Magnética/métodos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapiaRESUMO
BACKGROUND Renal podocyte damage plays a crucial role in the development of diabetic nephropathy. Genistein is derived from a leguminous plant, and MyD88 and TRIF are adaptor molecules in the Toll-like receptor (TLR) signaling pathway, which may play a role in autophagy. In this study, we utilized an in vitro high glucose (HG)-treated podocyte model to investigate the effects and underlying mechanisms of Genistein and MyD88 or TRIF siRNA induced autophagy and renal protection. MATERIAL AND METHODS An immortalized mouse podocyte cell line was treated with HG, Genistein, chloroquine, and/or transfected with specific Myd88 and TRIF siRNAs. The formation of autophagosomes and related autophagic vacuoles were monitored by transmission electron microscopy. The expression of autophagy-related factors and podocyte structure and functional markers, including LC3, p62, p-mTOR, synaptopodin, and nephrin, were measured by Western blot, and LC3 and p-mTOR expression were also assessed by immunofluorescence. RESULTS We showed that HG transiently (after 6-h exposure) induced expression of the autophagy activation marker LC3-II in podocytes. Genistein treatment induced autophagy in both normal and HG-treated podocytes through inactivating mTOR signaling. Moreover, Genistein protected podocytes against chloroquine in HG-cultured conditions in vitro by maintaining the level of autophagy-related proteins. In addition, MyD88 siRNA downregulated expression of autophagy-related proteins, whereas Genistein treatment reversed these effects. CONCLUSIONS This study demonstrated that Genistein-induced autophagy could be a potential treatment strategy for glomerular diseases.
Assuntos
Autofagia/efeitos dos fármacos , Genisteína/farmacologia , Glucose/toxicidade , Fator 88 de Diferenciação Mieloide/metabolismo , Podócitos/patologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Cloroquina , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: We sought to confirm that neutrophil gelatinase-associated lipocalin (NGAL) protects against apoptosis during endotoxemia. METHODS: Endotoxemia was induced in rats with lipopolysaccharide (LPS; 3.5 mg/kg) and serum creatinine (SCr), urinary NGAL (uNGAL), renal histopathology confirmed acute kidney injury (AKI). Renal caspase 3 and NGAL were assayed with immunohistochemistry 6 h later. A HK-2 cell model was used in which NGAL and caspase 3 mRNA were evaluated by qRT-PCR within 6 h after LPS (50 µM) treatment, and correlations were studied. NGAL and caspase 3 mRNA expression were measured after delivering NGAL siRNA in HK-2 cells and apoptosis was measured with TUNEL and flow cytometry. RESULTS: SCr and uNGAL were significantly increased after LPS treatment and renal morphology data indicated AKI and renal tubular epithelial cell apoptosis. Caspase 3 and NGAL were predominantly expressed in the tubular epithelial cells and there was a correlation between caspase 3 and NGAL protein (r = 0.663, p = 0.01). In vitro, there was a strong correlation between caspase 3 and NGAL mRNA in LPS-injured HK-2 cells within 24 h (r = 0.448, p < 0.05). Suppressing the NGAL gene in HK-2 cells increased caspase 3 mRNA 4.5-fold and apoptosis increased 1.5-fold after LPS treatment. CONCLUSIONS: NGAL is associated with caspase 3 in renal tubular cells with endotoxin-induced kidney injury, and may regulate its expression and inhibit apoptosis.
Assuntos
Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Proteínas de Fase Aguda/biossíntese , Apoptose/fisiologia , Túbulos Renais/metabolismo , Lipocalinas/biossíntese , Lipopolissacarídeos/toxicidade , Proteínas Proto-Oncogênicas/biossíntese , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Humanos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Lipocalina-2 , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND Hyperglycemia is one of the most dangerous factors causing diabetic nephropathy. Salidroside is considered to have the effects of reducing oxidative stress damage and improving cell viability. This study was performed to investigate whether and how salidroside reduces high-glucose (HG)-induced apoptosis in mouse podocytes. MATERIAL AND METHODS We examined whether salidroside could decrease HG-induced podocyte oxidative stress and podocyte apoptosis in vitro. The potential signaling pathways were also investigated. Podocytes (immortalized mouse epithelial cells) were treated with normal glucose (5.5 mM) as control or HG (30 mM), and then exposed to salidroside treatment. RESULTS HG enhanced the generation of intracellular reactive oxygen species (ROS) and apoptosis in podocytes. Salidroside reduced HG-induced apoptosis-related consequences via promoting HO-1 expression. Salidroside increased the expression level of phosphorylated Akt (p-Akt) and phosphorylated ILK (p-ILK), p-JNK, and p-ERK and localization of Nrf-2. JNK inhibitor and ILK inhibitor decreased HO-1 expression to different degrees. Moreover, specific siRNAs of ILK, Nrf-2, and HO-1, and inhibitors of HO-1 and ILK significantly increased ROS generation and Caspase9/3 expression in the presence of salidroside and HG. CONCLUSIONS The results suggest that salidroside reduces HG-induced ROS generation and apoptosis and improves podocytes viability by upregulating HO-1 expression. ILK/Akt, JNK, ERK1/2, p38 MAPK, and Nrf-2 are involved in salidroside-decreased podocyte apoptosis in HG condition.
Assuntos
Glucose/administração & dosagem , Glucosídeos/farmacologia , Heme Oxigenase-1/metabolismo , Fenóis/farmacologia , Podócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucosídeos/metabolismo , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/metabolismo , Fosforilação , Podócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The apoptosis of renal tubular epithelial cells contributes to the pathogenesis of diabetic nephropathy. High glucose-induced mitochondrial oxidative stress is considered to be an important mediator for renal tubular cell apoptosis. Sirtuin3(Sirt3), a kind of mitochondria-localized nicotinamide adenine dinucleotide(NAD+)-dependent protein deacetylase, has been reported to regulate the generation of ROS in mitochondria through regulating acetylation level and activity of several key mitochondrial enzymes. In this study, we investigated the role of Sirt3 on high glucose-induced apoptosis in HK-2 cells. High glucose decreased the protein and mRNA expression of Sirt3 in a time-dependent manner, along with increased cell apoptosis in HK-2 cells. Furthermore, high glucose-induced oxidative stress and apoptosis were reversed by Sirt3 overexpression or antioxidant treatment. Meanwhile, we also found that overexpression of Sirt3 or antioxidant could regulate the activity of Akt/FoxO signaling pathway associated with cell apoptosis in diabetic nephropathy. In conclusion, our data suggest that Sirt3 overexpression antagonize high glucose-induced apoptosis by controlling ROS accumulation and ROS-sensitive Akt/FoxO signaling pathway in HK-2 cells.
Assuntos
Células Epiteliais/fisiologia , Glucose/farmacocinética , Túbulos Renais Proximais/fisiologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/metabolismo , Apoptose/fisiologia , Linhagem Celular , Células Cultivadas , Células Epiteliais/citologia , Humanos , Túbulos Renais Proximais/citologia , Taxa de Depuração MetabólicaRESUMO
The neuroprotective role of propofol in transient global and focal cerebral ischemia reperfusion (I/R) animal model has recently been highlighted. However, no studies have conducted to explore the relationship between mitochondrial fission/fusion and I/R injury under the intervention of propofol. Moreover, neuroprotective mechanism of propofol is yet unclear. Culturing primary hippocampal cells were subjected to oxygen-glucose deprivation and re-oxygenation (OGD/R) model, as a model of cerebral I/R in vitro. Methods CCK-8 assay was used to test the effect of propofol on cell viability. We examined the effect of propofol on mitochondrial ultrastructure and mitochondrial fission evoked by OGD/R with transmission electron microscopy and immunofluorescence assay. To investigate possible neuroprotective mechanisms, the authors then examined whether propofol could inhibit calcium-overload, calcineurin (CaN) activation and the phosphorylation of dynamin-related protein 1 (Drp1) during the period of OGD/R, as well as the combination of Drp1-ser 637 and fission 1 (Fis1) protein by immunofluorescence assay, ELISA and double-labeling immunofluorescence analysis. Finally, the expression of Drp1-ser 637 and Fis1, apoptosis inducing factor (AIF) and cytochrome C (Cyt C) were detected by western blot. When added in culture media during OGD period, propofol (0.1µM-50µM) could alleviate neurons injury and protect mitochondrial ultrastructure, meanwhile inhibit mitochondrial fission. Furthermore, the concentration of intracellular free Ca2+, CaN activition and the phosphorylation of Drp1-ser637 were suppressed, as well as the translocation and combination of Drp1-ser 637 and Fis1. The authors also found that the expression of Cyt C, AIF, Drp1-ser637 and Fis1 were down-regulated. Notably, high dose of propofol (100µM-200µM) were confirmed to decrease the survival of neurons based on results of cell viability. Propofol could inhibit mitochondrial fission and mitochondrial apoptotic pathway evoked by OGD/R in rat hippocampal neurons, which may be via depressing calcium-overload.
Assuntos
Hipocampo/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Propofol/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Glucose/deficiência , Hipocampo/fisiopatologia , Hipocampo/ultraestrutura , Hipóxia/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Obesity-induced kidney injury contributes to albuminuria, which is characterized by a progressive decline in renal function leading to glomerulosclerosis and renal fibrosis. Matrix metalloproteinases (MMPs) modulate inflammation and fibrosis by degrading a variety of extracellular matrix and regulating the activities of effector proteins. Abnormal regulation of MMP-12 expression has been implicated in abdominal aortic aneurysm, atherosclerosis, and emphysema, but the underlying mechanisms remain unclear. The present study examined the function of MMP-12 in glomerular fibrogenesis and inflammation using apo E(-/-) or apo E(-/-)MMP-12(-/-) mice and maintained on a high-fat-diet (HFD) for 3, 6, or 9 months. MMP-12 deletion reduced glomerular matrix accumulation, and downregulated the expression of NADPH oxidase 4 and the subunit-p67(phox), indicating the inhibition of renal oxidative stress. In addition, the expression of the inflammation-associated molecule MCP-1 and macrophage marker-CD11b was decreased in glomeruli of apo E(-/-)MMP-12(-/-) mice fed HFD. MMP-12 produced by macrophages infiltrating into glomeruli contributed to the degradation of collagen type IV and fibronectin. Crescent formation due to renal oxidative stress in Bowman's space was a major factor in the development of fibrogenesis and inflammation. These results suggest that regulating MMP-12 activity could be a therapeutic strategy for the treatment of crescentic glomerulonephritis and fibrogenesis.
Assuntos
Gorduras na Dieta/efeitos adversos , Glomerulosclerose Segmentar e Focal/enzimologia , Glomérulos Renais/enzimologia , Macrófagos/enzimologia , Metaloproteinase 12 da Matriz/biossíntese , Obesidade/enzimologia , Animais , Gorduras na Dieta/farmacologia , Modelos Animais de Doenças , Fibrose , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Glomérulos Renais/patologia , Macrófagos/patologia , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologiaRESUMO
BACKGROUND: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and is associated with excessive cardiovascular morbidity and mortality. The angiotensin converting enzyme inhibitor (ACEI) benazepril has been shown to slow the progression of chronic renal disease and have beneficial effects in patients with a combination of chronic renal disease and cardiovascular disease. Transforming growth factor-ß(1) (TGF-ß(1)) plays a central role in the pathogenesis and progression of DN. Integrin-linked kinase (ILK) can modulate TGF-ß(1)-induced glomerular mesangial cell (GMC) injury, which is a prominent characteristic of renal pathology in kidney diseases. As an integrin cytoplasmic-binding protein, ILK regulates fibronectin (FN) matrix deposition and the actin cytoskeleton. Smooth muscle α-actin (α-SMA) is involved in progressive renal dysfunction in both human and experimental renal disease. METHODS: To explore the mechanisms of benazepril's reno-protective effects, we examined the expression of TGF-ß(1), ILK, and α-SMA in GMC exposed to high glucose (HG) and in the kidneys of streptozotocin (STZ)-induced diabetic rats using real-time quantitative RT-PCR and western blot analysis. To elucidate the mechanism(s) of the effect of benazepril on GMC cellular processes, we assessed the effect of benazepril on Angiotensin II (Ang II) signalling pathways using western blot analysis. RESULTS: The expression of TGF-ß(1), ILK, and α-SMA increased significantly in the diabetic group compared with the control group. Benazepril treatment inhibited the expression of these genes in DN but failed to rescue the same levels in the control group. Similar results were found in GMC treated with HG or benazepril. Ang II increased ERK and Akt phosphorylation in the HG group, and benazepril could not completely block these responses, suggesting that other molecules might be involved in the progression of DN. Our findings suggest that benazepril decreases ILK and α-SMA expression, at least in part, by affecting the interactions between Ang II and TGF-ß(1). CONCLUSIONS: The findings described here support the hypothesis that the HG milieu of diabetes increases TGF-ß(1) secretion, which increases the synthesis of ILK and α-SMA that are involved in the progression of DN. This might be an important mechanism of the benazepril renal-protective function in the pathogenesis of DN.
Assuntos
Actinas/biossíntese , Benzazepinas/uso terapêutico , Diabetes Mellitus Experimental/metabolismo , Células Mesangiais/metabolismo , Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Actinas/antagonistas & inibidores , Animais , Benzazepinas/farmacologia , Células Cultivadas , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Regulação da Expressão Gênica , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/patologia , Músculo Liso/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
BACKGROUND: Gremlin, a bone morphogenetic protein antagonist, plays an important role in the pathogenesis of diabetic nephropathy (DN). However, the specific molecular mechanism underlying Gremlin's involvement in DN has not been fully elucidated. In the present study, we investigated the role of Gremlin on cell proliferation and accumulation of extracellular matrix (ECM) in mouse mesangial cells (MMCs), and explored the relationship between Gremlin and the ERK1/2 pathway. METHODS: To determine expression of Gremlin in MMCs after high glucose (HG) exposure, Gremlin mRNA and protein expression were evaluated using real-time polymerase chain reaction and western blot analysis, respectively. To determine the role of Gremlin on cell proliferation and accumulation of ECM, western blot analysis was used to assess expression of pERK1/2, transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF). Cell proliferation was examined by bromodeoxyuridine (BrdU) ELISA, and accumulation of collagen IV was measured using a radioimmunoassay. This enabled the relationship between Gremlin and ERK1/2 pathway activation to be investigated. RESULTS: HG exposure induced expression of Gremlin, which peaked 12 h after HG exposure. HG exposure alone or transfection of normal-glucose (NG) exposed MMCs with Gremlin plasmid (NG + P) increased cell proliferation. Transfection with Gremlin plasmid into MMCs previously exposed to HG (HG + P) significantly increased this HG-induced phenomenon. HG and NG + P conditions up-regulated protein levels of TGF-ß1, CTGF and collagen IV accumulation, while HG + P significantly increased levels of these further. Inhibition of Gremlin with Gremlin siRNA plasmid reversed the HG-induced phenomena. These data indicate that Gremlin can induce cell proliferation and accumulation of ECM in MMCs. HG also induced the activation of the ERK1/2 pathway, which peaked 24 h after HG exposure. HG and NG + P conditions induced overexpression of pERK1/2, whilst HG + P significantly induced levels further. Inhibition of Gremlin by Gremlin siRNA plasmid reversed the HG-induced phenomena. This indicates Gremlin can induce activation of the ERK1/2 pathway in MMCs. CONCLUSION: Culture of MMCs in the presence of HG up-regulates expression of Gremlin. Gremlin induces cell proliferation and accumulation of ECM in MMCs. and enhances activation of the ERK1/2 pathway.
Assuntos
Matriz Extracelular/metabolismo , Glucose/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células Mesangiais/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Citocinas , Matriz Extracelular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Mesangiais/citologia , Células Mesangiais/efeitos dos fármacos , CamundongosRESUMO
AIM: To observe the effects of megsin gene transfection on the expressions of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1(ICAM-1). METHODS: Mouse glomerular mesangial cells were cultured in high glucose medium, and then cell proliferation was measured by MTT assay at 12, 24 and 48 h respectively. The expressions of megsin, MCP-1 and ICAM-1 in mesangial cells were detected by immunocytochemical staining and Western blotting. The concentration of type IV collagen in the culture supernatant of mesangial cells was measured by ELISA. RESULTS: Under high glucose concentration, the expressions of megsin, MCP-1 and ICAM-1 increased; the concentration of type IV collagen in the cell supernatant was elevated as well; and mesangial cell proliferation was enhanced. After megsin gene transfection, the above changes were more significant, but were abated following megsin shRNA/transfection. CONCLUSION: Megsin gene can up-regulate the expressions of MCP-1 and ICAM-1, promote mesangial cell proliferation and mesangial extracellular matrix accumulation.
Assuntos
Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica , Molécula 1 de Adesão Intercelular/metabolismo , Células Mesangiais/metabolismo , Serpinas/genética , Serpinas/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Colágeno Tipo IV/metabolismo , Mesângio Glomerular/metabolismo , Camundongos , TransfecçãoRESUMO
BACKGROUND: Neutrophil gelatinase associated lipocalin (NGAL) is a highly predictive biomarker of acute kidney injury. To understand the role of NGAL in renal injury during sepsis, we investigated the temporal changes and biological sources of NGAL in a rat model of acute kidney injury, and explored the relationship between renal inflammation, humoral NGAL and NGAL expression during endotoxemia. METHODS: To induce acute renal injury, rats were treated with lipopolysaccharide (LPS, 3.5 mg/kg, ip), and the location of NGAL mRNA was evaluated by in situ hybridization. Quantitative RT-PCR was also used to determine the dynamic changes in NGAL, tumor necrosis factor α (TNFα) and interleukin (IL)-6 mRNA expression 1, 3, 6, 12, and 24 hours following LPS treatment. The correlation among NGAL, TNFα and IL-6 was analyzed. Urinary and plasma NGAL (u/pNGAL) levels were measured, and the relationship between humoral NGAL and NGAL expression in the kidney was investigated. RESULTS: Renal function was affected 3-12 hours after LPS. NGAL mRNA was significantly upregulated in tubular epithelia at the same time (P < 0.001). The course of NGAL mRNA upregulation occurred in parallel with renal damage. There was a transient increase in TNFα and IL-6 mRNA levels within 3 hours following LPS administration, and a strong correlation between TNFα and NGAL mRNA (r = 0.995, P <0.001) but not with IL-6 mRNA. Both pNGAL and uNGAL levels were markedly increased compared with those in the control group (P < 0.001); however, only uNGAL levels were correlated with NGAL mRNA (r = 0.850, P <0.001). CONCLUSIONS: NGAL upregulation is sensitive to LPS-induced renal TNFα increase and injury, which are observed in the tubular epithelia. Urinary NGAL levels accurately reflect changes in NGAL in the kidney.
Assuntos
Injúria Renal Aguda/metabolismo , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Proteínas de Fase Aguda/urina , Análise de Variância , Animais , Biomarcadores/sangue , Biomarcadores/urina , Creatinina/sangue , Expressão Gênica , Interleucina-6/metabolismo , Glomérulos Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Lipocalina-2 , Lipocalinas/sangue , Lipocalinas/urina , Lipopolissacarídeos , Masculino , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas/urina , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Regulação para Cima , Urotélio/metabolismoRESUMO
BACKGROUND: Diabetic nephropathy is a complex disease with poor outcomes, and our current treatment measures are limited. It is urgent to search for novel therapeutic targets. Recently, a mesangium-predominant gene, megsin, has emerged as a participant in mesangial cell proliferation and/or mesangial matrix expansion. This study investigated the effect of megsin down-regulation on the progression of diabetic nephropathy. METHODS: Streptozotocin (STZ)-induced diabetic CD-1 mice after uninephrectomy received a pBAsi mU6 Neo megsin siRNA plasmid for 12 weeks and were compared with age-matched nondiabetic mice. In vitro mouse mesangial cells were transfected with pBAsi mU6 Neo megsin siRNA plasmid using Lipofectamine 2000 reagent and further cultured in DMEM containing high glucose for up to 48 hours. All of the cells were collected for protein extraction and the supernatant for type IV collagen measurement. The expression of megsin, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteases-2 (TIMP-2) and p27(Kip1) was determined by Western blotting. RESULTS: The megsin siRNA plasmid alleviated proteinuria and glomerular type IV collagen accumulation 12 weeks after the STZ injection, down-regulated renal cell proliferation and normalized the imbalance between MMP-2 and TIMP-2. Also, in vitro experiments showed that the glomerular mesangial cellular proliferation and type IV collagen production induced by high glucose were relieved after the transfection of megsin siRNA plasmid. The level of p27(kip1) was down-regulated in transfected mesangial cells significantly. CONCLUSIONS: The study suggests that the down-regulation of megsin might exert beneficial effects on the diabetic kidney partly through down-regulation of p27(kip1) level and that megsin may serve as a novel therapeutic target in the management of diabetic nephropathy.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Diabetes Mellitus Experimental/terapia , Nefropatias Diabéticas/prevenção & controle , Terapia Genética/métodos , Células Mesangiais/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Serpinas/genética , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Colágeno Tipo IV/metabolismo , Creatinina/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Regulação para Baixo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Células Mesangiais/patologia , Camundongos , Nefrectomia , Proteinúria/etiologia , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/prevenção & controle , RNA Interferente Pequeno/administração & dosagem , Serpinas/metabolismo , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , TransfecçãoRESUMO
Diabetic nephropathy is a complex and poorly understood disease process, and our current treatment options are limited. It remains critical, then, to identify novel therapeutic targets. Recently, a developmental protein and one of the bone morphogenetic protein antagonists, Gremlin, has emerged as a novel modulator of diabetic nephropathy. The high expression and strong co-localization with transforming growth factor-beta1 in diabetic kidneys suggests a role for Gremlin in the pathogenesis of diabetic nephropathy. We have constructed a gremlin siRNA plasmid and have examined the effect of Gremlin inhibition on the progression of diabetic nephropathy in a mouse model. CD-1 mice underwent uninephrectomy and STZ treatment prior to receiving weekly injections of the plasmid. Inhibition of Gremlin alleviated proteinuria and renal collagen IV accumulation 12 weeks after the STZ injection and inhibited renal cell proliferation and apoptosis. In vitro experiments, using mouse mesangial cells, revealed that the transfect ion of gremlin siRNA plasmid reversed high glucose induced abnormalities, such as increased cell proliferation and apoptosis and increased collagen IV production. The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid. Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels. We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy.